Compositions and methods relating to prevention of chemotherapy-induced alopecia

ABSTRACT

The present invention relates to a method for protecting a human patient or a mammalian animal to be subjected to chemotherapy treatment of a tumor not residing in the scalp of the patient or the skin of the animal against chemotherapy-induced alopecia, comprising administering to the scalp of the patient or the skin of the animal an effective amount of a composition comprising a chemical inducer of the stress protein response sufficiently prior to the administration of a chemotherapeutic drug. It also relates to pharmaceutical compositions for the prevention of chemotherapy-induced alopecia. It further relates to a method for protecting a human patient or a mammalian animal to be subjected to chemotherapy treatment of a tumor not residing in the scalp of the patient or the skin of the animal against chemotherapy-induced alopecia, comprising administering to the scalp of the patient or the skin of the animal an effective heat dose sufficiently prior to the administration of a chemotherapeutic drug.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/939,161, filed Aug. 24, 2001 which is a continuation-in-part ofinternational application PCT/IB01/00422 designating the United States,filed Mar. 21, 2001, which application claims priority to U.S.provisional application 60/191,580, filed Mar. 23, 2000.

FIELD OF THE INVENTION

The invention relates to conditions and compositions capable of inducingthe stress response in lair follicles and to methods of using saidconditions and compositions for prevention of chemotherapy-inducedalopecia.

BACKGROUND

Chemotherapy frequently induces hair loss. With chemotherapy, patientsnot only experience reduced stamina and independence but also must weara physical symbol of their illness in the loss of their hair. This lossof hair is a traumatic experience that may well result in lowerself-esteem and overall resistance. Some patients are known to haverefused chemotherapy for fear of losing their hair. Scalp tourniquetshave been used for several decades to prevent chemotherapy-inducedalopecia. This technique involves the placement of a pneumatictourniquet around the hairline at the time of administration of thechemotherapeutic drug. The tourniquet is then inflated to a pressureabove the systolic arterial pressure, reducing blood flow to the scalp.The effectiveness of this technique has never been unambiguouslydemonstrated. The use of tourniquets has more or less been replaced byscalp hypothermia. With this technique, the scalp temperature is loweredto below 24° C. by application of cold packs, etc., prior tochemotherapy. The technique has been reported to afford a 50-70% good toexcellent hair protective effect. However, results have remainednotoriously variable. Furthermore, the practice is rather uncomfortableand is only tolerated for a short time. It is likely to be mosteffective for chemotherapy agents with short half lives. Moreover,several cases of scalp metastases in patients who used scalp hypothermiawere reported. Finally, the technique appears to work considerably lesswell for combination chemotherapy than for therapy using single agents.Several pharmacological approaches for the prevention ofchemotherapy-induced hair loss were also tested. For a review, see Dorr.1998. Semin. Oncol. 25: 562-570. Most of the drugs tested failed (forexample, alpha-tocopherol, minoxidil, calcitriol) or showed a marked sexpreference (1,25-dihydroxyvitamin D3). More promising results wereobtained with the immunomodulatory substance ammonium trichloro(dioxy-ethylene-0,0′) tellurate (AS101). Sredni et al. 1996. Int. J.Cancer 65: 97-103. However, confirmation of this study is still beingawaited. Furthermore, the question has to be resolved whether theimmunomodulator is only effective if administered weeks prior tochemotherapy. If so, that would diminish somewhat the usefulness of thecompound. Another drug candidate may be ImuVert, perhaps used incombination with acetylcysteine. ImuVert is a membrane vesicle-ribosomepreparation from Serratia marescens. The combination of AS101 andacetylcysteine showed efficacy in a rodent model, but no human data areavailable. Some caution may be appropriate, since Imuvert as abiological response modifier has the potential of producing unacceptabletoxicities. Thus, there is no drug on the market that generally protectsagainst chemotherapy-induced alopecia, and there are only few drugcandidates that are under active development. There is therefore a needfor additional drug candidates and methods for protection againstchemotherapy-induced alopecia.

SUMMARY OF THE INVENTION

Cells, tissues, organs and entire organisms respond to proteotoxicstress by enhancing the expression of a set of proteins that are termedheat shock or stress proteins (Hsps). This response is referred toherein as the stress protein response, and conditions and compounds thatelicit this response are referred to as inducers. Conditions that elicitthe response are specifically referred to as physical inducers, andcompounds that elicit the response as chemical inducers. Based on whatis currently known about the likely consequences of activation of thestress protein response in cancerous cells, tissues and organs, it isimportant to avoid activation of this response during chemotherapeutictreatment of cancer. The present invention is based on the realizationby the inventor that there exists a particular situation, in which theprotective effects of elevated levels of Hsps can be harnessed toprevent treatment-unrelated toxicity of chemotherapeutic drugs withoutcompromising the efficacy of the same drugs viz-a-viz a tumor. Thisspecific situation relates to the scalp hair of a patient undergoingchemotherapy treatment of a tumor not residing in the scalp or to thefur coat or parts thereof of a mammalian animal subjected tochemotherapy of a tumor not located in the skin. Many chemotherapeuticdrugs and combinations of such drugs cause hair loss (alopecia) from thepatient's scalp or from the animal's fur coat. A chemical inducer of thestress protein response can be applied to the scalp of a patient or tothe skin of an animal such that it reaches the mitotically active cellsof the hair follicles before entering the general circulation. As aconsequence, the hair follicle cells and, depending on the nature of thecomposition comprising the chemical inducer, some other cells of theskin can be exposed to a concentration of chemical inducer that issufficiently high to activate the stress protein response in thesecells. Levels of stress proteins will increase, and, as a consequence,hair follicle cells will be protected against subsequent exposure tocytotoxic chemotherapeutic agents for a period of typically from 1-2days, and the alopecia phenotype will not develop. While, inevitably, afraction of chemical inducer molecules will eventually enter the generalcirculation, because of the high degree of dilution of chemical inducerin the circulation and because the stress protein response is notactivated before a threshold concentration of chemical inducer isattained, activation of the stress response will be limited to cells ofthe hair follicles and, possibly, skin cells and will not occur to asignificant extent in cells of the blood or other organs. Thus,topically administered chemical inducer will only activate theprotective stress protein response in hair follicles and, possibly, inthe skin but not elsewhere in the body and, consequently, will notnegatively affect the efficacy of chemotherapy treatment of tumors notlocated in the scalp or skin. In the case of a chemotherapeutic regimein which a chemotherapeutic drug is only administered once, a singletopical pretreatment of the patient or animal with a chemicalinducer-comprising composition may suffice to produce the hairfollicle-saving effect. Many chemotherapy regimes involve several cyclesof treatment with chemotherapeutic drug which cycles may be days orweeks apart. In these cases topical administration of a compositioncomprising a chemical inducer can be similarly periodical, precedingeach cycle of treatment with chemotherapeutic drug. With this type ofregime, chemical inducer will be eliminated during each treatment cycleand will never accumulate to a level sufficient for systemic activationof the stress protein response.

Thus, the invention relates to a method for protecting a human patientor a mammalian animal to be subjected to chemotherapy treatment of atumor not residing in the scalp of the patient or the skin of the animalagainst chemotherapy-induced alopecia. Protecting a human patient or amammalian animal comprises preventing or reducing the severity ofchemotherapy-induced alopecia. The method comprises administering to thescalp of the patient or the skin of the animal an effective amount of acomposition comprising a chemical inducer of the stress proteinresponse. Administration of chemotherapeutic drug is delayed for asufficiently long time to permit induction of the stress proteinresponse to take place and stress proteins in hair follicles toaccumulate to protective levels. An effective amount of a compositioncomprising a chemical inducer is an amount that is at least equal to theamount required to cause a measurable increase in the concentration ofat least one stress protein from the group of Hsps including Hsp90,Hsp70, Hsp25-27 and P-glycoprotein in hair follicles residing in skinexposed to the chemical inducer-comprising composition and that producesan increased resistance of the hair follicles to chemotherapeutic drugs.A measurable increase in the concentration of an Hsp is an increase ofat least 25% over the concentration measured prior to administration ofa composition of the invention. Exposure of cultured cells to a chemicalinducer typically results in a rapid increase in Hsp expression and in asufficient increase in Hsp concentrations within 2 to 12 hours to rendercells resistant against toxicants including chemotherapeutic drugs.However, skin including hair follicles represents a significant barrier,and additional time, up to 24 hours, can be required for a chemicalinducer to reach an effective concentration in hair follicle cells.Hence, chemotherapeutic drug is preferably administered between 2 and 36hours after administration to the scalp of a patient or to the skin ofan animal of a composition comprising a chemical inducer of the stressprotein response. More preferably, administration of chemotherapeuticdrug is delayed by 8 to 24 hours. Many chemical inducers of the stressprotein response are known. Generally, any compound that produces somemeasure of proteotoxicity functions as a chemical inducer. Preferredinducers are compounds of the benzoquinone ansamycin series (e.g.,geldanamycin), arsenic salts (e.g., sodium arsenite), tin salts (e.g.,stannous chloride), zinc salts (e.g., zinc chloride) and diamide. Afurther preferred chemical inducer is an activated heat shocktranscription factor 1 (HSF1) that may be administered as a recombinantprotein or as a nucleic acid containing a gene for the factor in anexpressible form.

The method of the invention also encompasses pretreatment of the scalpof a patient or the skin of an animal with compositions that comprise achemical inducer and additionally a penetration enhancer to facilitatetransport of inducer to the cells of the hair follicles.

The invention also relates to pharmaceutical compositions for protectionagainst chemotherapy-induced alopecia, the compositions comprising achemical inducer of the stress protein response, a penetration enhancerand an appropriate diluent or solvent. Preferred chemical inducers usedin these compositions are diamide, compounds of the benzoquinoneansamycin series, arsenic salts, tin salts, zinc salts and activated HSFin protein or nucleic acid form.

The invention further relates to the use of a chemical inducer of thestress protein response for the manufacture of a medicament forprotecting a human patient or a mammalian animal to be subjected tochemotherapy treatment of a tumor not residing in the scalp of thepatient or the skin of the animal against chemotherapy-induced alopecia,an effective amount of which medicament is administered to the scalp ofthe human patient or the skin of the mammalian animal sufficiently priorto administration of chemotherapeutic drug. An effective amount of suchmedicament is an amount that is at least equal to the amount required tocause a measurable increase in the concentration of at least one stressprotein from the group of Hsps including Hsp90, Hsp70, Hsp25-27 andP-glycoprotein in hair follicles residing in skin exposed to thechemical inducer-comprising medicament and that produces an increasedresistance of the hair follicles to chemotherapeutic drugs. A measurableincrease in the concentration of an Hsp is an increase of at least 25%over the concentration measured prior to administration of a medicamentof the invention. Preferably, chemotherapeutic drug is administeredbetween 2 and 36 hours after administration to the scalp of a patient orto the skin of an animal of a medicament comprising a chemical inducerof the stress protein response. More preferably, administration ofchemotherapeutic drug is delayed by 8 to 24 hours. Many chemicalinducers of the stress protein response are known. Generally, anycondition or compound that produces some measure of proteotoxicityfunctions as an inducer. Preferred chemical inducers for use in themanufacture of a medicament of the invention are compounds of thebenzoquinone ansamycin series (e.g., geldanamycin), arsenic salts (e.g.,sodium arsenite), tin salts (e.g., stannous chloride), zinc salts (e.g.,zinc chloride) and diamide. An additional preferred chemical inducer isan activated heat shock transcription factor 1 (HSF1) that may beadministered as a recombinant protein or as a nucleic acid containing agene for the factor in an expressible form. Also encompassed by theinvention is the use of a chemical inducer of the stress proteinresponse and of a penetration enhancer facilitating delivery of inducerto hair follicles for the manufacture of a medicament for protectingagainst chemotherapy-induced alopecia.

The invention also relates to a method using a physical inducer of thestress protein response, e.g., heat, for protecting a human patient or amammalian animal to be subjected to chemotherapy treatment of a tumornot residing in the scalp of the patient or the skin of the animalagainst chemotherapy-induced alopecia. Protecting a human patient or amammalian animal comprises preventing or reducing the severity ofchemotherapy-induced alopecia. In one embodiment, the method comprisesadministering to the scalp of the patient or the skin of the animal aneffective heat dose. Administration of chemotherapeutic drug is delayedfor a sufficiently long time to permit induction of the stress proteinresponse to take place and stress proteins in hair follicles toaccumulate to protective levels. An effective heat dose is a dose atleast equal to the dose required to cause a measurable increase in theconcentration of at least one stress protein from the group of Hspsincluding Hsp90, Hsp70, Hsp25-27 and P-glycoprotein in hair folliclesresiding in skin exposed to the heat dose and that produces an increasedresistance of the hair follicles to chemotherapeutic drugs. A measurableincrease in the concentration of an Hsp is an increase of at least 25%over the concentration measured prior to administration of a compositionof the invention. Exposure of cultured cells to a heat dose typicallyresults in a relatively rapid increase in Hsp expression and in asufficient increase in Hsp concentrations within 2 to 24 hours to rendercells resistant against toxicants including chemotherapeutic drugs.Hence, chemotherapeutic drug is preferably administered between 2 and 24hours after administration to the scalp of a patient or to the skin ofan animal of a heat dose. More preferably, administration ofchemotherapeutic drug is delayed by 6 to 12 hours. Heat can beadministered by several different means. Contact of the scalp of apatient or the skin of an animal in need of treatment with a heatedsurface or with a heated liquid (e.g., water) will provide a heat doseto the skin and the hair follicle cells. Other means for heating skinand hair follicle cells include exposure to ultrasound, or to microwave,infrared or radiofrequency radiation.

Accordingly, the embodiments of the invention described herein alsorelate to a method for the treatment of cancer in a human patient or amammalian animal in need thereof, comprising (a) administering to thescalp of the patient or the skin of the animal an effective dose of aphysical inducer such as heat or an effective amount of a compositioncomprising a chemical inducer of the stress protein response and (b)subjecting said human patient or animal to chemotherapy treatment.

DETAILED DESCRIPTION

Hair consists of the hair root, the hair bulb (the germinative center)and the hair shaft. Cells proliferate in the hair bulb, and the hair ispushed from the root through the scalp. The final product is a strand oftightly compacted keratin. Hair growth occurs in three phases. The firstphase is the anagen phase, which is the growth phase. 85-90% of humanhair follicles are in the anagen phase. Each hair follicle comprises abulbous base of mitotically active matrix cells. From these all cells ofthe hair shaft differentiate and grow. Cells move up in rows to theupper bulb and elongate vertically. Finally, they are being forcedupwards and emerge at the skin surface. Human hair bulb cells divide onthe average every 12 to 24 hours. Because of this substantial mitoticactivity, the hair bulb cells are particularly susceptible to cytotoxicagents. The anagen phase lasts between two and six years in humans. Thesecond stage is the catagen phase, which lasts a few weeks in humans. Inthis phase the hair root is separated from the hair bulb, pigmentstorage is terminated, and the root end is pushed out from the bulb.Less than 1% of human hair is in the catagen phase. The third phase isthe telogen phase, which is characterized by a lack of mitotic activity.This phase lasts between three and six months. About 10% of human hairis in the telogen phase. Dorr. 1998. Semin. Oncol. 25: 562-570. Hussein.1993. South. Med. J. 86: 489-496.

Alopecia or hair loss is frequently associated with cancer chemotherapy.Dorr. 1998. Semin. Oncol. 25: 562-570. Many of the commonly usedchemotherapeutic drugs induce hair loss, although there appear to bedifferences in the ability of different drugs to cause alopecia. Mostsevere effects are produced by cyclophosphiamide, daunorubicin,docetaxel, doxorubicin, etoposide, ifosfamide, paclitaxel, teniposideand topotecan. Joss et al. 1988. Recent Res. Cancer Res. 108: 117-126.Perry (ed). The Chemotherapy Source Book, Baltimore, Md., Williams &Wilkins, 1996, pp. 293-555, 595-606. Somewhat less effective in inducinghair loss are actinomycin, 5-fluorouracil, hydroxyurea, methotrexate,mitomycin, mitoxantrone, nitrogen mustard, vinblastine, vincristine,vindesine and vinorelbine. Oftentimes, these cytotoxic, chemotherapeuticdrugs are used in combination, which increases the risk of alopecia overthat inherent in the individual drugs.

There has been relatively little research to identify the actualmechanism(s) of chemotherapy-induced alopecia. Presumably, this is dueto the fact that the hypothesis that cytotoxic agents kill hair folliclecells by the same mechanism by which they kill cancer cells and otherproliferating cells is immediately plausible. Nevertheless, doxorubicinwas shown to kill hair cells by setting off an apoptotic mechanism.Cece. 1996. Lab. Invest. 75: 601-609. The same study also discoveredthat the targets of doxorubin toxicity were matrix and upper bulb cellsof the hair follicle. Another study reported that cyclophosphamideinduced massive apoptosis in anagen hair follicles. Schilli et al. 1998.J. Invest. Dermatol. 111: 598-604.

Theoretically, there would appear to be several ways to preventchemotherapy-induced hair loss, namely (1) reduction of the amount ofchemotherapeutic agent delivered to the bulb, (2) local inactivation ofthe chemotherapeutic drug, and (3) protection of bulb cells as proposedby the invention disclosed herein. The present invention relates todeliberate localized induction of the stress protein response in thescalp of a patient or the skin of a mammalian animal in need ofchemotherapy to protect hair follicles against the cytotoxic effects ofchemotherapeutic agents and combinations thereof without compromisingthe therapeutic efficacy of the latter agents.

Cells in every organ and every tissue respond to proteotoxic stress byenhancing the expression of so called heat shock or stress proteins(Hsps). This response is being referred to herein as the stress proteinresponse. For reviews, see Voellmy. 1994. Crit. Rev. Eukaryotic GeneExpr. 4: 357-401. Voellmy. 1996. In: Stress-Inducible Cellular Responses(Feige et al. eds.), Birkhauser Verlag, Basel, Switzerland, pp. 121-137.Parsell and Lindquist. 1993. Annu. Rev. Genet. 27: 437-496.Historically, the term “Hsp” was used to describe those proteins whoserates of synthesis were increased in cells exposed to the prototypicstressor heat. Hsps were distinguished based on their subunit molecularweights. Major Hsps have subunit sizes of about 110, 90, 70, 60, 20-30,and 10 kDa, respectively, and are referred to as Hsp110, Hsp90, Hsp70,Hsp60, Hsp20-30 (or small Hsp) and Hsp10, respectively. It is now knownthat most of these Hsps are molecular chaperones that assist folding andrefolding of proteins, intracellular trafficking of proteins, assemblyand dissociation of protein complexes, protein degradation, etc. Stressproteins are also known to participate in the regulation of the activityand stability of important cellular regulatory proteins such as steroidhormone receptors, certain signaling kinases including Raf and Ras, andtelomerase. In agreement with their physiological functions, Hsps arenot only prevalent in stressed cells but also in unstressed cells.Certain Hsps are major proteins even in the unstressed cell. Forexample, Hsp90 represents 1-2% of total cellular protein in the absenceof stress. When cells are stressed, concentrations of Hsps increasefurther.

It was long known that most Hsps are encoded by families of highlyrelated genes. While some of these genes are strictly stress-regulated,others are already substantially active in the unstressed cell. Some ofthe genes are not stress-regulated at all and express stress protein atall times. The latter genes are also referred to as cognate stressprotein genes, and the proteins encoded by them as stress or heat shockcognate proteins (Hscs as opposed to Hsps). The best known family ofstress protein genes encodes proteins with subunit molecular weights ofabout 70 kDa (Hsp/c70). Humans possess an hsp70 gene that is alreadysubstantially active in the unstressed cell, and whose activity isincreased by about 10 fold during heat stress. This gene is also knownas the hsp70A gene. There are at least two other genes, referred to ashsp70B and hsp70B′ genes, that are strictly heat-regulated. Theiractivity increases by about 1000 fold in the heat-stressed cell. Humancells also have at least one hsc70 gene encoding a protein that ishighly related to Hsp70. This gene is essentially not stress-regulated.

As discussed before, the activity of stress-regulatable hsp genes isincreased when the cell is exposed to a proteotoxic stress. Suchproteotoxic stress may be induced, for example, by heat, UV light,electromagnetic field, heavy metal ions such as a Cd, Zn, Sn, or Cuions, other sulfhydryl-reactive compounds such as sodium arsenite (anarsenic salt), inhibitors of energy metabolism, in particular inhibitorsof mitochondrial function, amino acid analogs such as canavanine orazetidine carboxylate, protein denaturants such as ethanol, oxidizingagents such as diamide (diazinedicarboxylic acid bis(N,N-dimethylamide))or other agents including, for example, toxicants that form proteinadducts such as acetaminophen. The activity of hsp genes is alsoelevated in cells exposed to inhibitors of proteolysis such aslactacystin or to compounds that interfere with the proper function of astress protein. Examples for the latter type of compound are thebenzoquinone ansamycins including geldanamycin and herbimycin A that areknown to specifically bind Hsp90 in its nucleotide-binding site. Thecurrent model that appears to be generally accepted in the field holdsthat exposure to any of these stresses results in an increased rate ofprotein unfolding and, consequentially, in an elevated concentration ofnonnative protein. A sufficiently elevated level of nonnative proteintriggers increased expression of hsp genes. Quantitative measurementssuggested that substantially increased hsp gene activity requiresdenaturation of about 1-2% of cellular protein. Because exposure to theabove chemicals or physical conditions results in increased hsp geneactivity, these chemicals or physical conditions are also referred to aschemical or physical inducers of the stress protein response. Chemicalas well as physical inducers can be used for the practice of the presentinvention.

The stress regulation of hsp genes is mediated by a heat shocktranscription factor (HSF). Mammalian cells express several differentbut related HSF molecules. Only one of these factors, HSF1, appears tobe normally involved in the stress regulation of hsp genes. HSF1 is aubiquitously expressed factor that is inactive, i.e., incapable oftransactivating an hsp gene, in the unstressed cell. When the cell isexposed to one of the above-described inducers, the factor is activatedand acquires transactivation ability. In the unstressed cell, HSF1 formspart of a dynamic heterooligomeric complex that includes Hsp90 and,possibly, other chaperones and co-factors. Zou et al. 1998. Cell 94:471-480. When the cell is stressed, nonnative proteins accumulate. Thesenonnative proteins bind preferentially Hsp90 and other chaperones,competing with HSF1 for binding the same chaperones. As a result of thiscompetition, a fraction of HSF1 is no longer chaperone-bound.Unassociated HSF1 rapidly homotrimerizes and, as a consequence, acquiresthe ability to specifically bind so called heat shock element (HSE)sequences present in promoters of hsp genes. It appears that for fullactivation HSF1 further needs to be hyperphosphorylated. Recentunpublished observations raise the possibility that activatingphosphorylation events may be negatively regulated by binding ofchaperone complexes to the trimeric transcription factor.

Mutagenesis studies of human HSF1 led to the discovery of mutant factorsthat are no longer stress-regulated but are capable of transactivatinghsp genes in the absence of any stress. Zuo et al. 1995. Mol. Cell.Biol. 15: 4319-4330. Xia et al. 1999. Cell Stress & Chaperones 4: 8-18.These mutant factors that function as chemical inducers of the stressprotein response are also referred to herein as activated HSF1.Deletions and amino acid substitutions in the region between about aminoacids 185 and 315 of the 529-residue-long human HSF1 polypeptide resultin this deregulated phenotype. Deletions and substitutions in the regionbetween about amino acids 200 and 315 are known to be constitutivelytransactivating when overexpressed from transfected genes. Of particularinterest are substitutions and deletions in the region between aboutamino acids 185 and 200 which yield factors that are constitutivelyactive even at exceedingly low concentrations. Examples of deletions andsubstitutions known to render HSF1 constitutively transactivating weredescribed in patent application PCT/US98/01038 (WO98/31803) which isincorporated herein in its entirety by reference. It is noted thatapplication WO98/31803 also described nonhuman HSF and chimeric factorscapable of transactivating hsp genes in the absence of stress. While notevery deletion or substitution in the residue-185-315 region will resultin a deregulated human HSF1, the identification of deregulated mutantfactors is readily achieved by a person skilled in the art, using one ofseveral methods of analysis. For example, a gene encoding a mutated HSF1to be tested may be inserted in a suitable expression vector. Theresulting expression construct may be introduced by transfection in acell containing one or more copies of an hsp promoter-driven reportergene. An example of such a cell line is HeLa-CAT, a human cell linecontaining several copies of a chloramphenicol acetyltransferase geneunder the control of a human hsp70B promoter. Baler. et al. 1992. J.Cell Biol. 117: 1151-1159. Increased reporter gene activity which can bemeasured by a convenient assay of reporter activity will indicate that amutated HSF1 is capable of transactivating an hsp gene in the absence ofstress.

Exposure of cells to a nonlethal heat stress was long known to protectthe cells against a subsequent more severe heat stress that is lethal tonaive cells. Parsell and Lindquist. 1993. Annu. Rev. Genet. 27: 437-496.Heat pretreatment also protects cells against certain chemical stresses.This protective effect is correlated with increased expression of Hsps.Transfection experiments provided direct evidence that increased levelsof certain individual stress proteins produce stress tolerance. Forexample, cells transfected to transiently overexpress Hsp70 or celllines stably overexpressing the same Hsp were found to have an increasedstress resistance. Li et al. 1991. Proc. Natl. Acad. Sci USA 88:1681-1685. Huot et al. 1991. Cancer Res. 51: 5245-5252. Jaattela et al.1992. EMBO J. 11: 3507-3512. Analogous observations were made in animalexperiments. The ability of Hsps to protect against ischemia/reperfusiondamage in the heart was demonstrated by heat preconditioning experiments(Liu et al. 1992. Circulation 86: 11358-11363. Richard et al. 1996.Fund. Clin. Pharmacol. 10: 409-415. Joyeux et al. 1998. Cardiovasc. Res.40: 124-130) as well as by studies using transgenic animals. In thelatter studies. hearts of transgenic mice overexpressing Hsp70 weresubjected to an ischemic event. Recovery of the hearts from ischemictrauma was assessed following 30 minutes of reperfusion after theischemic event. As judged from measurements of contractile force andcreatine kinase release, hearts from transgenic mice showed asignificant improvement of recovery when compared to hearts fromnon-transgenic animals. Plumier et al. 1995. J. Clin. Invest. 95:1854-1860. Marber et al. 1995. J. Clin. Invest. 95: 1446-1456. Similarresults were obtained in experiments in which hearts of adult rats weretransfected with an hsp70 gene by intracoronary infusion of anHVJ-liposome formulation containing the hsp70 gene. Suzuki et al. 1997.J. Clin. Invest. 99: 1645-1650. Transgenic mice overexpressing Hsp70 inthe brain also exhibited reduced neural damage following middle cerebralartery occlusion. Plumier et al. 1997. Cell Stress & Chaperones 2:162-167. Preconditioning of rabbits with heat or a tin salt was found toprevent paralysis caused by acute spinal cord ischemia. Perdrizet et al.1999. Ann. N.Y. Acad. Sci. 874: 320-325. Personal communication.Similarly, protection of kidney function from ischemic damage wasdemonstrated in a pig model. Perdrizet et al. 1999. Ann. N.Y. Acad. Sci.874: 320-325.

Regarding protective effects of stress proteins in the skin, it wasdemonstrated repeatedly that heat preconditioning increases the survivalof skin flaps. This enhanced survival correlated with increasedexpression of Hsp70 in the skin flaps. Koenig et al. 1992. Plast.Reconstr. Surg. 90: 659-694. Wang et al. 1998. Plast. Reconstr. Surg.101: 776-784. Furthermore, heat preconditioning protected keratinocyteand epithelial cell cultures against UVB-induced damage. This protectiveeffect was associated with elevated Hsp levels, in particular Hsp70levels. Trautinger et al. 1995. J. Invest. Dermatol. 105: 160-162.Injection of an Hsp70 antibody increased the sensitivity ofkeratinocytes to UVB injury. Bayerl and Jung. 1999. Exp. Dermatol. 8:247-253.

Cells expressing a constitutively active HSF1 mutant overexpressed Hspsand exhibited increased resistance to heat stress, simulated ischemiaand exposure to cyclophosphamide (tested in hepatocyte-derived (HepG2)cells). Xia et al. 1999. Cell Stress & Chaperones 4: 8-18.Overexpression of stress protein Hsp70 enhanced cellular resistance toadriamycin. Roigas et al. 1998. Prostate 34: 195-202. Overexpression ofHsp27 also resulted in resistance to doxorubicin. Richards et al. 1996.Cancer Res. 56: 2446-2451. Oesterreich et al. 1993. Cancer Res. 53:4443-4448. Karlseder's laboratory and others similarly reported thatspecific overexpression of Hsp70 or Hsp27 protected cells againstdoxorubicin-induced apoptosis. Karlseder et al. 1996. Biochem. Biophys.Res. Commun. 220: 153-159. Richards et al. 1996. Cancer Res. 56:2446-2451. Oesterreich et al. 1993. Cancer Res. 53: 4443-4448. Hsp70 orHsp27 overexpression also rendered cells resistant to cisplatin.Komatsuda et al. 1999. Nephrol. Dial. Transplant. 14: 1385-1390.Richards et al. 1996. Cancer Res. 56: 2446-2451. Oesterreich et al.1993. Cancer Res. 53: 4443-4448. These studies demonstrated clearly thatincreased expression of individual Hsps results in protection ofparticular cell types from the toxicity of cytotoxic chemotherapeuticagents. Because of the conserved structure and function of stressproteins and the conservation of the stress protein response, it isexpected that the latter findings similarly apply to other cell typesthan those studied as well as to cells in tissues. It is furtherexpected that overexpression of Hsps will also protect cells againstother cytotoxic agents than those tested in the above studies and thatoverexpression of the entire cohort of Hsps will have at least acomparable protective effect than overexpression of individual Hsps.Finally, several studies supported the notion that activation of thestress protein response also induces multidrug resistance. Chin et al.1990. J. Biol. Chem. 265: 221-6. Kim et al. 1998. Exp. Mol. Med. 30:87-92. These findings suggest that activation of the stress proteinresponse will diminish the efficacy of cytotoxic chemotherapeutic drugsused alone or in combination in cancer chemotherapy. Thus, activation ofthe stress protein response during cancer chemotherapy treatment isclearly counterindicated.

The protective effect of an activated stress protein response on cancercells may be diminished somewhat by other mechanisms. Continuedoverexpression of a constitutively active HSF1 inhibited cell growth.Xia et al. 1999. Cell Stress & Chaperones 4: 8-18. Growth-arrested cellsmay be less susceptible to cytotoxic agents than growing cells. However,it appeared that growth arrest of activated HSF1-overexpressing cellswas due to the effective redirection of these cells towards productionof excessive amounts of Hsps in lieu of other essential proteins. It isdoubtful that this situation is physiologically relevant. Hsps have aprivileged relationship with the immune system. In the late 1980s, anumber of investigators realized that Hsps were preferred targets forhumoral and cellular immune responses to infection by bacteria, fungiand protozoa. These findings were puzzling because stress proteins evenfrom divergent organisms are highly related. Hence, autoimmune reactionsmay occur. Indeed, infected, vaccinated and even healthy patientsexpress antibodies and T-cells directed against stress proteins.Apparently, immune responses against stress proteins are finely tuned,and severe autoimmune reactions are avoided. More recently it wasdiscovered that stress proteins drastically enhance the immunogenicityof covalently and non-covalently linked antigens. Interestingly, andthis distinguishes stress proteins from most other adjuvants, stressprotein-enhanced immunity appears to be predominantly of a Th1-liketype, stimulating phagocytes and activation of cytotoxic lymphocytes(CTL). Huang et al. 2000. J. Exp. Med., in press. While the underlyingmechanism for the immunological activity of stress proteins is not wellunderstood, it is suspected that it may involve stimulation of antigenpresentation. Over the last few years, several studies were publishedsuggesting that increased expression of stress proteins alone mayenhance presentation by tumor cells of their antigens and, hence, maystimulate immune responses directed against the tumor cells. Melcher etal. 1998. Nat. Med. 4: 581-587. Todryk 1999. J. Immunol. 163: 1398-1408.Wells et al. 1997. Scand. J. Immunol. 45: 605-612. However, whileanti-tumor activity of preparations containing stress proteins complexedwith antigenic peptides/proteins could be demonstrated in tumor models,the importance of effects affecting the immune system resulting fromoverexpression of stress proteins within tumor cells remains uncertain.It seems unlikely that the latter effects would be capable of cancelingout the cytoprotective effects of overexpressed stress proteins, whichcytoprotective effects will diminish the efficacy of chemotherapytreatment.

Thus, based on what is currently known about the likely consequences ofactivation of the stress protein response in cancerous cells, tissuesand organs, it is critically important to avoid activation of the stressprotein response during chemotherapy treatment of cancer. The presentinvention is based on the realization by the inventor that in at leastone particular situation it is possible to harness the protectiveactivity of elevated levels of Hsps to prevent treatment-unrelatedtoxicity of chemotherapeutic drugs without compromising the efficacy ofthe drugs viz-a-viz the cancer in need of chemotherapy treatment. Thissituation concerns the hair follicles in the scalp of a cancer patientor in the skin of an animal in need of chemotherapy. As discussedbefore, treatment-unrelated toxicity of many chemotherapeutic drugs andcombinations of drugs results in loss of scalp hair in a human patientand in loss of hair from the fur coat of treated animals. A chemicalinducer of the stress protein response can be administered directly tothe scalp of a cancer patient or the skin of an animal such that itreaches the mitotically active cells of the hair follicles prior toentering circulation, i.e., without much dilution. Levels of stressproteins in inducer-exposed hair follicle cells and, possibly, someother cells of the skin will increase, and, within a few hours, hairfollicles will be protected against subsequent exposure to cytotoxicchemotherapeutic agents for a period of typically from 1-2 days.Eventually, a fraction of the inducer molecules will enter the bloodstream. However, because of the high level of dilution of chemicalinducer in the blood stream, and because chemical inducer needs toattain a threshold concentration before a stress protein response ismounted, activation of the stress protein response will remain limitedto cells of the hair follicles and, possibly, of the skin and will notoccur to a significant extent in cells of the blood or other organs.Hence, chemical inducer will never reach but a negligible systemicconcentration, which concentration is too low to affect the efficacy ofchemotherapy treatment of tumors not residing in hair follicles or, iftopically administered chemical inducer is not specifically targeted tohair follicles, in skin exposed to inducer. Because chemotherapy regimesfrequently involve several cycles of administration of chemotherapeuticdrugs days or weeks apart, administration of chemical inducer can alsobe periodical, preceding each cycle of administration ofchemotherapeutic drugs. Even if administered repeatedly, with this typeof administration regime chemical inducer will be eliminated during eachtreatment cycle and will never accumulate to levels sufficient forsystemic activation of the stress protein response. Thus, the presentinvention involves the topical administration of an effective amount ofa chemical inducer of the stress protein response to the scalp of acancer patient or the skin of an animal sufficiently prior to theadministration of a chemotherapeutic agent to treat a cancer notresiding in inducer-exposed cells to selectively activate a protectivestress protein response in the scalp of the patient or the skin of theanimal. A chemical inducer may also be topically administered to anyother region of the human body susceptible to chemotherapy-inducedalopecia, such as for example eyebrow, beard and mustache regions.Furthermore, it is also expected that the methods and compositions ofthe invention will also be effective for the protection against alopeciacaused by radiation treatment. Thus, the invention also encompasses anyof the embodiments described for the protection of a human patient oranimal from radiation-induced alopecia. As used herein, an “effectiveamount” refers to amount of a chemical inducer (or inducer-comprisingcomposition) that will elicit the biological response of hair folliclesof a human patient or animal or the medical response of a human patientor animal that is being treated by a researcher or clinician. The term“effective amount” comprises any amount which, as compared to acorresponding hair follicle-containing tissue or human or animal subjectwhich has not received such amount, results in increased resistance ofhair follicles against killing by chemotherapeutic agents or in improvedtreatment, prevention, or severity reduction of chemotherapy-inducedalopecia.

Alternatively, a physical inducer of the stress protein response such astransient heat can be targeted directly to the scalp of a cancer patientor the skin of an animal such that it reaches the mitotically activecells of the hair follicles but does not penetrate much below the skin.Levels of stress proteins in inducer-exposed hair follicle cells andother cells of the skin will increase, and, within a few hours, hairfollicles will be protected against subsequent exposure to cytotoxicchemotherapeutic agents for a period of typically from 1-2 days. Becauseof the targeted administration of the physical inducer, stress proteinlevels will not increase in other cells than skin cells, and theefficacy of chemotherapy treatment of tumors not residing in hairfollicles or other skin locations will not be diminished. Becausechemotherapy regimes frequently involve several cycles of administrationof chemotherapeutic drugs days or weeks apart, administration ofphysical inducer can also be periodical, preceding each cycle ofadministration of chemotherapeutic drugs. Thus, the present inventionalso involves the targeted administration of an effective dose of aphysical inducer of the stress protein response to the scalp of a cancerpatient or the skin of an animal sufficiently prior to theadministration of a chemotherapeutic agent to treat a cancer notresiding in inducer-exposed cells to selectively activate a protectivestress protein response in the scalp of the patient or the skin of theanimal. A physical inducer may also be targeted to any other region ofthe human body susceptible to chemotherapy-induced alopecia, such as forexample eyebrow, beard and mustache regions. Furthermore, it is expectedthat this embodiment of the methods of the invention will also beeffective for the protection against alopecia caused by radiationtreatment. Thus, the invention also encompasses any of the embodimentsdescribed for the protection of a human patient or animal fromradiation-induced alopecia. As used herein, an “effective dose” refersto a dose of a physical inducer that will elicit the biological responseof hair follicles of a human patient or animal or the medical responseof a human patient or animal that is being thought by a researcher orclinician. The term “effective dose” comprises any dose which, ascompared to a corresponding hair follicle-containing tissue or human oranimal subject which has not received such dose, results in increasedresistance of hair follicles against killing by chemotherapeutic agentsor in improved treatment, prevention, or severity reduction ofchemotherapy-induced alopecia.

Inducers

As discussed before, inducers of the stress protein response includephysical inducers such as heat, UV radiation, electromagnetic field andchemical inducers such as heavy metal ions, e.g., Cd, Zn, Sn or Cu ions,other sulfhydryl-reactive compounds, e.g., sodium arsenite (an arsenicsalt), inhibitors of energy metabolism, in particular inhibitors ofmitochondrial function, amino acid analogs, e.g., canavanine orazetidine carboxylate, protein denaturants, e.g., ethanol andguanidinium hydrochloride, oxidizing agents, e.g., diamide, and otheragents, e.g., toxicants that form protein adducts such as acetaminophen.Inducers also include inhibitors of proteolysis such as lactacystin andcompounds that interfere with the proper function of an Hsp. Examples ofthe latter type of compound include benzoquinone ansamycins such asgeldanamycin and herbimycin A that are known to specifically bind Hsp90in its nucleotide-binding site. For a list of typical inducers see Zouet al. 1998. Cell Stress & Chaperones 3: 130-141. The above list is notexhaustive. Many additional chemicals are also known to be inducers ofthe stress protein response. Some of these chemicals including biclomol,cyclopentenones and certain prostaglandins do not appear to fit into anyof the above-cited groups. Furthermore, there is little doubt that newchemical inducers will be discovered in the future, because, generally,any compound that has some degree of proteotoxicity will induce thestress protein response. Whether a particular compound will beproteotoxic may or may not be readily deduced from its structure. Itseems therefore more appropriate to define chemical inducersfunctionally rather than structurally. For the purposes of thisinvention an inducer is a compound that is capable of enhancing Hspexpression at a sublethal concentration or is a sublethal physicalcondition that stimulates Hsp expression. There are many methods fordiscovering whether or not a compound/physical condition is an inducer.For example, parallel mammalian cell cultures can be exposed to a rangeof sublethal concentrations of a substance to be tested in the presenceof a radiolabeled amino acid. After an appropriate exposure period,cells are harvested and lysed, and cell lysates are subjected toSDS-PAGE and autoradiography or fluorography. If the substance tested isa chemical inducer, it will enhance the rate of synthesis ofpolypeptides with molecular weights typical for Hsps (e.g., 90, 70,25-27 kDa), In a more rigorous version of the same test, a particularHsp is immunoprecipiated from the cell lysates using an anti-Hspantibody, and the relative rate of synthesis of the Hsp is estimatedfrom SDS-PAGE and autoradiography or fluorography of immunoprecipitatedprotein. Anti-Hsp antibodies are commercially available, for example,from StressGen Biotechnologies Corp. of Victoria, B.C.

Note that not only small molecule compounds such as those discussedbefore are chemical inducers of the stress protein response. Chemicalinducers also include larger molecules such as proteins and nucleicacids. Nonlimiting examples of such chemical inducers are functionalgenes encoding a constitutively active HSF1 as well as constitutivelyactive HSF1 proteins. Their delivery to cells will induce stress proteinexpression that can be detected by the test described before. Alsoincluded are genes for individual stress proteins such as Hsp90, Hsp70,Hsp25-27 and P-glycoprotein and the proteins encoded by these genes.Their delivery to cells will partially reproduce the stress proteinresponse, i.e., result in an increased level of a particular stressprotein that can be detected by the above test.

Embodiments of the present invention involve topical administration of acomposition comprising a chemical inducer of the stress protein responseto the scalp of a cancer patient or the skin of a mammalian animal.Because of this mode of administration, the systemic concentration ofchemical inducer remains low. Consequently, there is relatively littledanger of systemic or organ-specific toxicity caused by a chemicalinducer. It would therefore appear that essentially any chemical inducercan be used in the compositions of the invention. Most preferred,however, will be chemical inducers that have already been tested or usedin humans such as, for example, tin salts, zinc salts and arsenic salts,or chemical inducers that are about to be tested in humans such as, forexample, a benzoquinone ansamycin. Also preferred are chemical inducerswith well known chemical reactivity such as diamide as well as chemicalinducers that are expected to be highly specific activators of thestress protein response such as an activated form of HSF1 delivered asnucleic acid or protein.

Formulations Comprising a Chemical Inducer and Delivery

Depending on its chemical properties (e.g., lipophilicity, molecularsize), a chemical inducer may be topically administered in a solvantsuch as ethanol, propylene glycol or glycerol. Schilli et al. 1998. J.Invest. Dermatol. 111: 598-604. Tata et al. 1994. J. Pharm. Sci. 83:1508-1510. Sredni et al. 1996. Int. J. Cancer 65: 97-103. Moretypically, a chemical inducer will be administered in a formulation thatalso includes one or more penetration enhancers (or promoters). Dermaland intrafollicular delivery are highly active fields of academic andindustrial research, and a person skilled in these arts will know ofappropriate methods for delivering a particular chemical inducer. Theterm “penetration enhancer (or promoter)” is used here in its broadestsense to include any physical method or any chemical composition thatincreases the permeability of the skin by temporarily compromising theintegrity and physicochemical properties of the skin or that results inselective targeting of hair follicles. It is also meant to includedelivery vehicles such as liposomes, including deformable andultradeformable liposomes, as well as active electric methods such asiontophoresis, ultrasonic vibration and electroporation. It alsoincludes the preparation of lipophilic derivatives of molecules to bedelivered. For example, tape stripping was used to enhance thepermeability of skin, particularly to macromolecules. Yang et al. 1995.Br. J. Dermatol. 133: 679-685. Repeated brushing of skin permittedefficient delivery even of naked DNA into the outer layers of theepidermis and hair follicles. Yu et al. 1999. J. Invest. Dermatol. 112:370-375. Well known chemical penetration enhancers are Azone, DegammaE,or n-decylmethyl sulphoxide. Hoogstraate et al. 1991. Int. J. Pharm. 76:37-47. Bodde et al. 1989. Biochem. Soc. Trans. 17: 943-945. Choi et al.1990. Pharm. Res. 7: 1099-1106. See also Marjukka Suhonen et al. 1999.J. Controlled Release 59: 149-161. Recent examples of chemicalpermeation enhancers are N-acetylprolinate esters, polyethyleneglycol-8-glyceryl caprylate/caprate, SEPA and hydrogels such asdeoxycholate-hydrogels. Tenjarla et al. 1999. Int. J. Pharm. 192:147-158. Tran. 1999. J. Surg. Res. 83: 136-140. Diani et al. 1995. SkinPharmacol. 8: 221-228. Valenta et al. 1999. Int. J. Pharm. 185: 103-111.Lipophilic derivatization of molecules to be delivered has beensuccessful, for example, in the case of IFNalpha. Acyl derivatives(chain length 12-16) showed much increased cutaneous and percutaneousabsorption than the underivatized molecule. Foldvari et al. 1999.Biotechnol. Appl. Biochem. 30: 129-137. Iontophoresis is a method basedon electrical stimulation of skin permeability for mostly ionizedmolecules. It has been used successfully to deliver in the skin smallmolecules as well as small polypeptides. Guy. 1998. J. Pharm. Pharmacol.50: 371-374. One of the latest electrical methods is electroporationthat has been used to deliver hydrophilic compounds in the skin. Bangaand Prausnitz. 1998. Trends Biotechnol. 16: 408-412. Methods fordelivering nucleic acids to hair follicles are also available. WO00/24895 and WO 98/46208.

The use of encapsulation technologies for skin delivery and,specifically, intrafollicular delivery of active molecules has become apreferred approach in recent years. A study by Fresta and Puglisisuggested that stratum corneum lipid-based unilamellar liposomes may besuitable devices for dermal delivery of drugs. Fresta and Puglisi. 1996.J. Drug Target 4: 95-101. Of great interest is the recent development ofultradeformable liposomes that have been used to deliver a variety ofsmall and large molecules to the skin. For example, vesicles containingphosphatidylcholine mixed with edge activators such as sodium cholate,Span 80 and Tween 80 were successfully used for the delivery of thehormone oestradiol. El Maghraby et al. 2000. Int. J. Pharm. 196: 63-74.Cevc. 1996. Crit. Rev. Ther. Drug Carrier Syst. 13: 257-388.Particularly relevant are findings that cationic lipid-basedformulations can deliver small and large molecules includingoligonucleotides to the hair follicles. This delivery may have exquisitespecificity since it takes place via the junction of the internal andexternal root sheath. Lieb et al. 1997. J. Pharm. Sci. 86: 1022-1029.Hoffman showed that phosphatidylcholine-based liposomes can target dyes,melanins, genes and proteins selectively to hair follicles. Hoffman.1998. J. Drug Target 5: 67-74. Genes delivered are active in thefollicle, making the follicle a target for selective gene therapy. Liand Hoffman. 1995. Nat. Med. 1: 705-706. Hoffman. 2000. Nat. Biotechnol.18: 20-21.

Dosage and Administration of Chemical Inducer

In the practice of the present invention, a composition comprising achemical inducer is applied to the scalp of a patient or the skin of anonhuman mammal prior to exposure of the patient or the mammal to acytotoxic, chemotherapeutic agent. In order to protect hair folliclecells against killing by the chemotherapeutic agent, the chemicalinducer must reach a concentration in the hair follicles that issufficiently high to activate the stress protein response in thefollicle cells, which results in an objectively measurable increase inthe concentration of at least one stress protein selected from the groupconsisting of Hsp90, Hsp70, Hsp25-27 and P-glycoprotein. Morepreferably, the levels of several or all of these stress proteins areelevated. An increase of about 25% in the concentration of a stressprotein is readily detectable by western blot analysis using an antibodyagainst the stress protein. While the ranges of concentrations thatcause a detectable stress protein response in mammalian cell culturesare known for many chemical inducers (see, for example, Zou et al. 1998.Cell Stress & Chaperones 3: 130-141, incorporated herein by reference)and can serve as an initial guide for dose-finding studies, theconcentrations required in compositions for topical administration tothe scalp of a patient (or skin of another mammal) are preferablydetermined empirically for each composition. It will be appreciated thatthe inducer concentration reached in the hair follicles is dependent onthe chemical properties of the inducer and on the efficacy of the chosenpenetration enhancer, and can be determined for each chemical inducerand penetration enhancer by the skilled person as further describedherein or by any other method known in the art. Standard clinicaldose-finding studies may be carried out to predict by how much levels ofstress proteins in hair follicles need to be increased for maximalprotection of the cells against various chemotherapeutic drugs. The mostrelevant clinical parameter to be measured is hair density before andafter chemotherapy. These measurements may be quantitative (hair countin an area of skin of defined size) or semiquantitative (estimatinggrades of alopecia). Alternatively or additionally, skin biopsies may betaken and analyzed for density and/or morphology of hair follicles. Asan imperfect substitute endpoint (see before) activation of the stressprotein response in hair follicle cells prior to administration ofchemotherapeutic drug can be estimated in scalp biopsies byimmunocytochemical methods (Hashizume et al. 1997. Int.J.Dermatol. 36:587-592. Yu et al. 1999. J.Invest.Dermatol. 112: 370-375) or westernblot using a stress protein antibody.

The time at which a composition comprising an inducer is bestadministered to the scalp of a patient (or skin of another mammal)relative to the time of initiation of a chemotherapy treatment cycle mayalso be determined empirically according to standard protocols. Kineticsof delivery of chemical inducer to the hair follicles will vary with thenature of the chosen inducer and penetration enhancer. In cell culture,exposure to a sufficient concentration of a chemical inducer results ina rapid activation of the stress protein response, and cytoprotectivelevels of stress proteins are reached within about 2-12 hours. As skinrepresents a significant barrier to delivery of molecules, attainment ofcytoprotective levels of stress proteins in hair follicles may bedelayed by up to 24 hours, depending on the nature of the chosenchemical inducer and penetration enhancer. Thus, a compositioncomprising a chemical inducer of the stress protein response may beadministered between about 2 and 36 hours prior to administration of achemotherapeutic agent. Preferably, a composition comprising a chemicalinducer of the stress protein response will be administered betweenabout 8 and 24 hours ahead of chemotherapy. Once cytoprotective levelsof stress proteins are reached in the cells of the hair follicles, thehair follicles will retain an increased resistance to chemotherapeuticagents for typically 1-2 days. With this guidance, a person skilled inthe art is enabled to empirically define with only routineexperimentation an appropriate dosage and an appropriate regime ofadministration of a particular composition comprising a chemical inducerthat provide effective protection of hair follicles againstchemotherapeutic agents.

Dosage and Administration of Physical Inducer

In another aspect of the practice of the present invention, the scalp ofa patient or the skin of a nonhuman mammal is exposed to a physicalinducer of the stress protein response prior to exposure of the patientor the mammal to a cytotoxic, chemotherapeutic agent. In order toprotect hair follicle cells against killing by the chemotherapeuticagent, the dose of physical inducer administered must be sufficientlyhigh to activate the stress protein response in the follicle cells,which results in an objectively measurable increase in the concentrationof at least one stress protein selected from the group consisting ofHsp90, Hsp70, Hsp25-27 and P-glycoprotein. More preferably, the levelsof several or all of these stress proteins are elevated. An increase ofabout 25% in the concentration of a stress protein is readily detectableby western blot analysis using an antibody against the stress protein. Apreferred physical inducer is heat. Heat may be delivered or produced ina target tissue by different means including direct contact with aheated surface or a heated liquid, ultrasound, infrared radiation, ormicrowave or radiofrequency radiation. For the practice of theinvention, a preferred means of delivering heat to the scalp of apatient or the skin of a mammal involves direct contact with a heatedliquid such as water. In a nonlimiting example, a patient is provided adevice resembling a shower cap that covers the scalp of the patient. Thecap extends slightly beyond the hairline of the patient and forms awatertight seal with the skin immediately adjacent to the hairline. Theinside of the cap contains an appropriate volume of water or otherphysiological aequous solution that is in correspondance with atemperature-controlled waterbath by means of an appropriate inlet andoutlet, valves, connecting tubes and a water pump. The range of heatdoses that cause a detectable stress protein response in mammalian cellcultures is known and can serve as an initial guide for dose-findingstudies. The typical range of elevated temperatures extends from about39° C. to about 45° C., and the typical duration of elevated temperatureexposures is between about 2 hours and 15 min. The appropriate heatdoses to be applied to the scalp of a patient (or skin of anothermammal) are preferably determined empirically. Standard clinicaldose-finding studies may be carried out to predict by how much levels ofstress proteins in hair follicles need to be increased for maximalprotection of the cells against various chemotherapeutic drugs. The mostrelevant clinical parameter to be measured is hair density before andafter chemotherapy. These measurements may be quantitative (hair countin a area of skin of defined size) or semiquantitative (estimatinggrades of alopecia). Alternatively or additionally, skin biopsies may betaken and analyzed for density and/or morphology of hair follicles. Asan imperfect substitute endpoint (see before) activation of the stressprotein response in hair follicle cells prior to administration ofchemotherapeutic drug can be estimated in scalp biopsies byimmunocytochemical methods (Hashizume et al. 1997. Int. J. Dermatol. 36:587-592. Yu et al. 1999. J. Invest. Dermatol. 112: 370-375) or westernblot using a stress protein antibody.

The time at which an appropriate heat dose is best administered to thescalp of a patient (or skin of another mammal) relative to the time ofinitiation of a chemotherapy treatment cycle may also be determinedempirically according to standard protocols. In cell culture, exposureto an appropriate heat dose results in a relatively rapid activation ofthe stress protein response, and cytoprotective levels of stressproteins are reached within hours rather than days. Thus, an appropriateheat dose may be administered between about 2 and 24 hours prior toadministration of a chemotherapeutic agent. Preferably, the heat dosewill be administered between about 6 and 12 hours ahead of chemotherapy.The latter time delays refer to initiation of chemotherapy treatmentafter initiation of heating. Once cytoprotective levels of stressproteins are reached in the cells of the hair follicles, the hairfollicles will retain an increased resistance to chemotherapeutic agentsfor typically 1-2 days. With this guidance, a person skilled in the artis enabled to empirically define with only routine experimentation anappropriate heat dose and an appropriate regime of administration of theheat dose that provide effective protection of hair follicles againstchemotherapeutic agents.

Animal Models of Chemotherapy Induced Alopecia

While imperfect stand-ins for the human patient, animal models ofalopecia can be used to evaluate inducers and protection methods. Humanhair growth appears to differ from that of many animals, in that inhumans 90% of follicles are in the anagen phase, whereas in adultanimals such as rodents this percentage is drastically lower. Two animalmodels that, with respect to growth phase, approach the human situationare newborn (8-day-old) rats (Hussein et al. 1990. Science 249:1564-1566) and C57/BL/6 mice after depilation of a portion of the furcoat. Paus et al. 1990. Br. J. Dermatol. 122: 777-784. Paus et al. 1994.Am. J. Pathol. 144: 719-734. In the first model, advantage is taken ofthe active phase of hair growth in the newborn rats, and in the secondmodel, hair regrowth is synchronized by depilation. In the mouse model,resting (telogen) hair follicles in the depilated skin of 6-8-week-oldfemale C57BL/6 mice are induced to enter active hair growth (anagen).This is achieved by painting the entire back or a desired portion of thefur coat of anesthesized animals (30 mg/kg pentobarbital) with a wax androsin mixture, which mixture is peeled off after hardening. Paus et al.1990. Br. J. Dermatol. 122: 777-784. Schilli et al. 1998. J. Invest.Dermatol. 111: 598-604. Pharmacological compositions typically areadministered topically about 5 days after depilation, at which time allhair follicles are in anagen III-IV of the hair cycle. Hence, aformulation containing a chemical inducer of the stress proteinresponse) or a dose of a physical inducer such as heat will beadministered at the latter time point. The two models were usedextensively in studies of alopecia induced by chemotherapeutic drugs,including adriamycin and cyclophosphamide. Balsari et al. 1994. FASEB J.8: 226-230. Schilli et al. 1998. J. Invest. Dermatol. 111: 598-604.Jimenez and Yunis. 1992. Cancer Res. 52: 413-415. The animal models maybe used for proof-of-principle experiments, for evaluation of potentialpenetration enhancers concerning their ability to improve delivery of achemical inducer to hair follicles, for estimation of the local toxicityof a chemical inducer, for a demonstration that localized delivery of achemical inducer or local exposure to a physical inducer does not resultin an elevated systemic concentration of the chemical inducer, ingeneralized activation of the stress protein response by the physical orchemical inducer, etc. The invention thus also comprises methods foridentifying agents (i.e., chemical inducers or combinations of chemicalinducers and penetration enhancers) for use in the protection of a humanor animal from chemotherapy-induced alopecia comprising (a)administering a test agent to an animal model of chemotherapy-inducedalopecia, and (b) determining whether said agent is capable of inducingthe stress protein response in said animal model. Also encompassed aremethods for identifying agents for use in the protection of a human oranimal from chemotherapy-induced alopecia comprising (a) selecting anagent capable of inducing the stress protein response, and (b)administering said test agent to an animal model of chemotherapy-inducedalopecia and determining whether said agent protects againstchemotherapy-induced alopecia.

To further illustrate the invention, nonlimiting examples of experimentsusing the above-mentioned animal models of alopecia are described in thesections that follow.

A chosen pharmacological treatment or physical treatment (e.g., heattreatment) will need to be shown to induce the stress protein responsein a majority of relevant cells of hair follicles. Further, it will beimportant for the optimization of a treatment regime to be able toassess the relative magnitude and duration of the induced stress proteinresponse in hair follicles. For these purposes, an immunohistochemicalassay will be utilized that estimates in cells of hair follicles andother cells of the skin levels of the major stress-inducible form ofHsp70. There are at least two valid reasons for the choice of inducibleHsp70 as a preferred indicator for the stress protein response. First,Hsp70 is one of the most abundant Hsps and was shown to be on its owncytoprotective. Liu et al. 1992. Cancer Res. 52: 3667-3673. Li et al.1995. Exp. Cell Res. 217: 460-468. Second, it is clear from scores ofprevious studies using cell lines and animal tissues that expression ofthe major inducible form of Hsp70 is tightly regulated in rodents. Welchet al. 1983. J. Biol. Chem. 258: 7102-7111. In the absence of stress,its level is very low to absent in all cell lines and most tissues.During and subsequent to stress, the protein rapidly accumulates to adramatically elevated level. A study by Hashizume et al. (Hashizume etal. 1997. Int. J. Dermatol. 36, 587-592) examined levels of inducibleHsp70 in the C57/BL/6 mouse and found that inducible Hsp70 expression inthe anagen hair follicles is low. Only during the anagen-catagentransformation did the level of inducible Hsp70 increase significantly.A monoclonal antibody that specifically detects inducible Hsp70 incultured rodent cells and in fresh and fixed tissue sections iscommercially available (<<C92>>, StressGen Biotechnologies Corp.,Victoria, BC (cat.#: SPA-810)).

Experiments to validate the immunohistochemical assay require thatexpression of Hsp70 is induced, since, as discussed before, this proteinis normally absent or only present at a very low level. Two differentmethods can be used for induction of the Hsp70. The first involvesexposing animals to whole body hyperthermia (by immersion in awaterbath). While optimal temperature and duration of the heat exposurewould need to be determined experimentally, previous cell cultureexperiments provide sufficient initial guidance for at least achieving,without further experimentation, a level of induction of Hsp70 that isdetectable immunohistochemically. The second method is based on previousobservations by Li and Hoffman. Li and Hoffman. 1995. Nat. Med. 1:705-706. These researchers found that liposomes containing a CMVpromoter-controlled β-galactosidase gene were efficiently andselectively delivering the β-galactosidase gene to mitotically activecells (matrix cells and presumptive follicle stem cells) of mouse anagenhair follicles, where the gene was actively expressed. The same protocolcan be used for introducing into follicle cells an expression constructfor an activated human HSF1 (mutant HSF1d202-316, referred tohereinafter as HSF1(+)). HSF1(+) is known to strongly enhance expressionof inducible Hsp70 in different cell types. Xia et al. 1999. Cell Stress& Chaperones 4: 8-18.

For assay validation experiments newborn rats and adult mice afterdepilation of a portion of their fur coat are exposed to moderatelysevere whole body hyperthermia. Alternatively or additionally, liposomescontaining a CMV promoter-driven hsp1(+) gene or, as a control, aβ-galactosidase gene are administered to areas on the backs of newbornrats or depilated areas of adult mice. After an appropriate time (6-48 hafter heat exposure, or 1, 3 or 5 days after transduction), treated anduntreated animals are sacrificed, and skin samples are taken. Thesesamples can be processed using a standard immunohistochemistry protocol.To provide an example protocol, the skin samples can be embedded inO.T.C. (Miles) and quick-frozen. Yu et al. 1999 J. Invest. Dermatol.112: 370-375. Frozen specimens can be sectioned on a cryostat (5 um) andcollected on clean, charged slides. Subsequent to air-drying andfixation in acetone, slides can be washed, blocked and exposed to Hsp70antibody C92. C92 antibody on the specimens can be detected with anappropriate enzyme-labeled secondary antibody. Alternatively, ifnecessary because of high background, a biotinylated C92 antibody(commercially available) may be used to eliminate the need for secondaryantibody.

It is noted that specific nucleic acid hybridization could be used as analternative assay of increased hsp70 gene expression in the unlikelyevent that antibody binding proves unsuccessful. Rat and mouse hsp70genes were cloned (Perry et al. 1994. Gene 146: 273-278. Longo et al.1993. J. Neurosci. 36: 325-335), and hybridization probes could,therefore, readily be prepared.

As was also discussed before, over the last ten years it became clearthat preferential and efficient delivery of small molecular weight drugsubstances as well as large molecules such as nucleic acids and proteinsto mitotically active cells of hair follicles can be achieved by topicaladministration of lipid-based formulations and liposomes containing theactive substance of interest. Balsari et al. 1994. FASEB J. 8: 226-230.Li and Hoffman. 1995. Nat. Med. 1: 705-706. Lieb et al. 1992. J. Invest.Dermatol. 99: 108-113. Lieb et al. 1997. J. Pharmaceutical Sciences 86:1022-1029. Li et al. 1993. In Vitro Cell. Dev. Biol. 29A: 192-194. Li etal. 1993. In Vitro Cell. Dev. Biol. 29A: 258-260. Li and Hoffman. 1995.In Vitro Cell. Dev. Biol. 31A: 11-13. Hoffman. 1997. J. Drug Targeting5: 67-74. Foldvari et al. 1999. Biotechnol. Appl. Biochem. 30: 129-137.For liposomes it was further shown that there is only negligible releaseof drug substance into the circulation. Balsari et al. 1994. FASEB J. 8:226-230. Li and Hoffman 1997. J. Derm. Sci. 14: 101-108. In the presentexample experiments liposomal formulations as described by the Hoffmangroup (Hoffman. 1997. J. Drug Targeting 5: 67-74) are used to deliver tohair follicle cells drug substances (chemical inducers) that induce thestress protein response. Hoffman's liposomes for small molecules andproteins were phosphatidylcholine-based, and those for nucleic acidscontained either phosphatidylcholine alone or phosphatidylcholinecholesterol: phosphatidylethanolamine in a 5:3:2 ratio.

In the example experiments two types of chemical inducers of the stressprotein response are tested (individually), small molecule compoundsodium arsenite and HSF1(+). HSF1(+) can be delivered as a nucleic acidencoding HSF1(+) or as recombinant protein. The nucleic acid can be aplasmid vector containing an hsf1(+) gene under the control of aconstitutively active cytomegalovirus (CMV) promoter. In a therapeuticsetting it will be desirable that the stress protein response is onlyinduced transiently. Although the plasmid-borne hsf1(+) gene will beinactivated with time, this inactivation may be considered too slow. Analternative would be to use a different (eukaryotic) expression vectorthat will allow for regulated expression of the hsf1(+) gene. Geneswitches that are activated/repressed by presumptively innocuous smallmolecular weight substances (e.g., tetracycline, RU486, etc.) are knownand are readily available. Gossen et al. 1996. Science 268: 1766-1769.Gossen and Bujard. 1992. Proc. Natl. Acad. Sci. USA 89: 5547-5551. Wanget al. 1997. Nat. Biotechnol. 15: 239. Wang et al. 1997. Gene Therapy 4:432-441. The latter issue does not arise if HSF1(+) is delivered as arecombinant protein. When wildtype human HSF1 and HSF1(+) were expressedfrom similar constructs in mammalian cells, wildtype HSF1 accumulated toa significantly higher level than HSF1(+) (unpublished data), suggestingthat the mutant protein (i.e., HSF1(+)) is considerably less stable thanthe wildtype protein. Subsequent experiments estimated the half life ofHSF1(+) to be 6-8 hours. Thus, introduction into cells of recombinantHSF1(+) can only produce a transient induction of the stress proteinresponse. HSF1(+) can be produced, for example, as a FLAG-tagged proteinor as a glutathione transferase fusion in E. coli. Voellmy. 1996. InStress-Inducible Cellular Responses, U. Feige, R. I. Morimoto, I.Yahara, and B. S. Polla, eds. (Basel: Birkhaeuser Verlag). pp. 121-137.Guo, Y., Guettouche, T., Fenna, M., Boellmann, F., Pratt, W. B., Toft,D. O., Smith, D. F., and Voellmy, R. Unpublished data. FLAG-taggedHSF1(+) and the glutathione transferase fusion protein can be purifiedby affinity chromatography methods. The glutathione transferase moietycan be cleaved off during purification, yielding HSF1(+). Because itdoes not prevent HSF1(+) function (unpublished result), removal of thetag from FLAG-tagged HSF1 may not be considered necessary. Essentiallypure recombinant proteins can be obtained. It is noted that because ofthe relative instability of HSF1(+) it will be advantageous to use aproduction strain that is low in proteolytic activity. While HSF1(+) maybe expressed in bacterial expression systems, it may also be expressedin and purified from eukaryotic expression systems, includingbaculovirus-infected insect cells.

Sodium arsenite is dissolved in water or phosphate-buffered saline at ornear maximal solubility. HSF1(+) protein or nucleic acid is dissolved atthe highest practical concentration. These solutions and series ofdilutions are then incorporated into liposomes as described by Hoffman.Hoffman. 1997. J. Drug Targeting 5: 67-74. Controls include emptyliposomes and liposomes containing a protein or nucleic acid unrelatedto HSF1(+), respectively. These liposomal preparations are administeredto areas on the back, side or abdomen of newborn rats (8-day-old) ordepilated areas of adult mice (5 days after depilation). Administrationmay be once or may be repeated at appropriate (e.g., daily) intervals.At different times (12 hours, 1-10 days) after the last administration,animals are sacrificed. Skin samples are taken, and sections areprepared and analyzed by the immunohistochemical assay described beforeas well as microscopically to estimate density and morphology of hairfollicles.

These experiments can answer several questions. Estimates can beobtained for each chemical inducer of the minimal and bestconcentrations to induce the stress protein response as well as of themaximal concentration at which inducer can be administered withoutcausing damage to hair follicles (only relevant for sodium arsenite).Regarding the latter information, the reader may be reminded that thestress protein response is induced in response to a marginal proteotoxicstress. Thus, at excessive concentrations a chemical inducer such assodium arsenite will have significant cytotoxicity and will kill hairfollicle cells. For this reason it is critical to determine ranges ofconcentrations at which the chemical inducer triggers Hsp overexpressionwithout causing irreversible damage. An excessive concentration ofinducer can be detected by a diminished stress protein response comparedto that induced by a lower concentration as well as by changes in themorphology and density of hair follicles.

Second, the experiments can show whether the liposomal preparationstarget all or nearly all hair follicle matrix cells (and putativefollicle stem cells). If inducer-containing liposomes prepared accordingto the directions provided by Hoffman (Hoffman. 1997. J. Drug Targeting5: 67-74) are found to target only a small fraction of mitoticallyactive matrix and putative stem cells, analogous experiments to thosedescribed above can be carried out to test liposomes of differentcomposition or other penetration enhancers.

Third, the experiments define, for each inducer, the time course ofactivation of the stress protein response as well as its persistence.This information is required for the design of effective alopeciaprevention regimes in the animal models. Optimal protection will onlyresult if chemotherapeutic drugs are administered after activation ofthe stress protein response occurred and Hsp concentrations increased toappropriately elevated levels. Note that the data obtained from theabove experiments only define a minimum delay between pretreatment withinducer and treatment with chemotherapeutic drug, i.e., they will onlyprovide initial conditions for the experiments described below. It willbe the latter experiments that define the level of inducible Hsp70 thatcorrelates with optimal protection against alopecia induced by achemotherapeutic agent. Data on the persistence of the stress proteinresponse allow for an estimation of whether one-time induction of theresponse is likely to provide protection for the entire period duringwhich a chemotherapeutic agent is expected to be present at an effectiveconcentration. In addition, they provide information on whether inducedlevels of Hsps persist for a sufficiently long time to be potentiallyprotective in animals subjected to regimes involving multipleadministration of a chemotherapeutic drug. Finally, they reveal whethersequential administration of several doses of inducer-containingliposomes effectively prolongs the period during which concentrations ofHsps are elevated.

Fourth, the experiments can also reveal whether repeated administrationof inducer-containing liposomes will, in addition to extending theduration of the stress protein response, produce a more pronouncedresponse and/or increase the fraction of matrix and putative stem cellsof hair follicles that mount a stress protein response.

Model Experiments for Establishing Conditions for Optimal Protection ofHair Follicles Against Selected Chemotherapeutic Agents

The following experiments can establish the conditions that result inoptimal protection of hair follicles against different chemotherapeuticagents in the animal models. Although chemotherapeutic agents arefrequently used in combination, animals will only be exposed to singledrugs in these experiments. Because questions relating to the relativeimportance of an individual drug in a particular combination areavoided, this simplification allows for a conclusive demonstration thatinduction of the stress protein response protects against hair follicletoxicity of a particular drug. The experiments described belowconcentrate on several drug substances that produce severe alopecia inhumans and that are present in many of the commonly used therapeuticcombinations. Selected chemotherapeutic drugs are cyclophosphamide,adriamycin, taxol, etoposide and vincristine.

In initial experiments, conditions are established under which singleintraperioneal injections of the different selected chemotherapeuticagents cause severe alopecia (grade 3, characterized by essentiallycomplete failure of hair growth/regrowth in most animals; see below).Previous studies can provide valuable guidance. For example, inductionof alopecia in newborn rats by adriamycin and cyclophosphamide wasdescribed by Hussein et al. (Hussein et al. 1990. Science 249:1564-1566), Jimenez and Yunis (Jimenez and Yunis. 1992. Cancer Res. 52:5123-5125), Balsari et al. (Balsari et al. 1994. FASEB J. 8: 226-230)and Jimenez et al. (Jimenez et al. 1995. Am. J. Med. Sci. 310: 43-47),and by etoposide by Davis et al. (Davis et al. 2001. Science 291:134-137). Alopecia in the C57/BL/6 mouse model resulting from exposureto cyclophosphamide was studied by Paus and collaborators. Paus et al.1994. Am. J. Pathol. 144: 719-734. Schilli et al. 1998. J. Invest.Dermatol. 111: 598-604. Other researchers described alopecia in micefollowing administration of adriamycin. Malkinson et al. 1993. J.Invest. Dermatol. 101: 135S-137S. D′Agostini et al. 1998. Int. J. Oncol.13: 217-224. To obtain results that are statistically meaningful, groupsconsisting of minimally ten animals for each data point are used inthese and subsequent experiments. The primary assay for alopecia ismacroscopic evaluation performed independently by two observers. Fourgrades are distinguished: grade 0: no alopecia, grade 1: mild alopeciadefined as less than 50% hair loss, grade 2: moderately severe alopeciadefined as more than 50% hair loss, and grade 3 with total or virtuallytotal (>90%) alopecia. Hussein et al. 1990. Science 249: 1564-1566.Sredni et al. 1996. Int. J. Cancer 65: 97-103. Corroboration of findingscan be obtained from microscopic examination of skin sections, whichexamination assesses density and morphology of hair follicles. Notethat, at least in the mouse model, increased pigmentation and skinthickness are known to be correlated with anagen progression (explainedin Paus et al. 1990. Br. J. Dermatol. 122: 777-784). Thus, should theneed arise, estimation of skin pigmentation and thickness could serve assubstitute assays of hair growth. These initial experiments define, foreach chemotherapeutic agent, the optimal concentration at whichvirtually complete alopecia is produced, the location on the animals'body in which the alopecia phenotype is most readily observed(Experiments will be conducted with mice depilated dorsally, laterallyand ventrally.), the time after administration at which expression ofthe phenotype is most readily evaluated as well as the reproducibilityof the expression of the phenotype. Note that in order to keepexperimental protocols as simple as possible, single administration ofchemotherapeutic agents is highly preferred.

To assess and optimize protective effects of an activated stress proteinresponse against alopecia induced by a chemotherapeutic agent, groups ofat least ten 8-day-old rats or C57/BL/6 mice 5 days after depilation aretreated topically (once or repeatedly as indicated) with liposomalpreparations containing a chosen inducer (here sodium arsenite andHSF1(+)) of the stress protein response. Three different preparationsare tested, the first containing inducer at the lowest concentration atwhich it triggers a measurable increase of the level of inducible Hsp70after 12 or 24 hours (as estimated in the experiments describedearlier), and the second and third containing successively higherconcentrations. Administration of a chemotherapeutic agent occurs either12 or 24 hours after (last) administration of inducer-containingliposomes or about 12 hours or 24 hours later. A predetermined amount ofa chemotherapeutic agent (defined in the preceding paragraph) isinjected intraperitoneally into all inducer-treated animals and a groupof mock-treated (with empty liposomes in experiments using sodiumarsenite or with liposomes containing a control protein or nucleic acidin experiments using HSF1(+) protein or gene) animals. Additionalinducer-and mock-treated groups are injected with vehicle only.Alternatively, to assess and optimize protective effects of an activatedstress protein response against alopecia induced by physical inducerheat, groups of at least ten 8-day-old rats or C57/BL/6 mice 5 daysafter depilation are treated topically (once or repeatedly as indicated)are subjected to local heat treatments of different intensity (heatexposure form 39 to 45° C. for 15 to 120 min) or are left untreated.Local heat treatment may be administered by several differentprocedures. A simple procedure may involve placing an anesthesizedanimal on an indented metal mesh fixed to a waterbath in such a way thatthe indented portion of the mesh and, consequently, the part of theanimal's body resting in this indented portion are immersed in water.Administration of a chemotherapeutic agent may occur either 12 or 24hours after (last) administration of a heat dose. A predetermined amountof a chemotherapeutic agent (defined in the preceding paragraph) isinjected intraperitoneally into all groups of animals.

Animals are then returned to quarters, and, at the time previouslyidentified as optimal for the assessment of the alopecia phenotype,grades of alopecia in all animals is recorded. The animals are thensacrificed, and skin samples are taken, fixed and sectioned. Sectionsare examined microscopically for hair follicle density and morphology.To confirm stress protein induction, several additional animals can beincluded in each group. These animals are sacrificed at the time ofadministration of the chemotherapeutic agent, and skin samples are takenand processed for immunohistochemical estimation of the level ofinducible Hsp70, i.e., of the degree of induction of the stress proteinresponse achieved.

For experiments in which alopecia is evaluated, individual animals areassigned an alopecia score ranging from 0 to 3 (see above). Alopeciascores for each treatment group are summarized by calculating the meanand standard deviation of alopecia scores of individual animals.Treatment groups are compared by one-way analysis of variance (ANOVA).If differences among treatment groups are detected by ANOVA, a post-hoctest (e.g., Scheffe's test or Student Newman Keuls test) can be used todetermine which groups are different from each other. The criterion forstatistical significance is a p<0.05.

To avoid possible systemic/organ toxicity of chemical inducers as wellas impairment of the therapeutic efficacy of chemotherapeutic agentswhich would result if the stress protein response were also induced incells of tumors to be treated, the above-described experiments usedtopically applied liposomal formulations to specifically deliverchemical inducers of the stress protein response to the relevant cellsof hair follicles. Because of this topical delivery, only the lattercells but not other cells including the tumor cells targeted by thechemotherapy treatment should be protected against toxicity from thechemotherapeutic agents. Based on the previous reports cited above,topical administration of liposomal formulations of chemical inducerscan be expected to result in the desired highly localized delivery ofthe inducers. Although small amounts of a chemical inducer such assodium arsenite may end up in the circulation, its concentration will beminimal due to dilution and, because it will be far below the requiredthreshold concentration, it will be incapable of activating the stressprotein response systemically. Similarly, the systemic concentration ofHSF1(+) is expected to be exceedingly low, and activation of the stressprotein response in blood cells and organs should occur at most in onlya few isolated cells. Note that the above discussion does not apply totreatment approaches in which a therapeutic stress protein response isinduced by localized heat treatment.

In an experiment aimed at ascertaining that topically administeredchemical inducers do not accumulate in the circulation and in majororgans to levels that are sufficient for the induction of the stressprotein response, animals are administered a liposomal formulationcontaining chemical inducer (sodium arsenite or a form of HSF1(+)) in anamount and under conditions known from previous experiments to beeffective in preventing alopecia caused by chemotherapeutic drugs and,after an appropriate delay, injected with a chemotherapeutic drug.Controls are animals treated similarly but with empty liposomes andliposomes containing a control protein or nucleic acid, respectively. Toestimate the contributions of the chemotherapeutic agent and the lipidcomponents of the liposomes to induction of the stress protein response,further controls can include animals that did not receivechemotherapeutic agent (vehicle-injected) or were not pretreated withchemical inducer-containing or control liposomes. At various times priorto and subsequent to the time of administration of chemotherapeuticdrug, animals are sacrificed and dissected. Extracts of PBL, heart,lung, brain, liver and kidney are prepared using routine methodology andare analyzed by western blot probed with antibody against inducibleHsp70 (C92). Levels of inducible Hsp70 are compared. As discussedbefore, this experiment is strongly expected to show induction of thestress response to be localized to cells of hair follicles.

The above disclosure cites numerous references. All publications,patents and patent applications cited herein are expressly incorporatedherein by reference. While the invention has been described herein withreference to specific features, aspects and embodiments, it will beappreciated that the scope of the invention is not thus limited, butrather extends to and encompasses other variations, modifications andother embodiments. Accordingly, the invention is to be correspondinglyinterpreted as including all such variations, modifications and otherembodiments within its spirit and scope as hereinafter claimed.

1. A method of reducing chemotherapy-induced alopecia in a human patientor a mammalian animal to be subjected to chemotherapy treatment of atumor not residing in the scalp or other region susceptible tochemotherapy-induced alopecia of the patient or the skin of themammalian animal against chemotherapy-induced alopecia comprising: a)administering a heat dose that causes an increase in the concentrationof at least one stress protein selected from the group consisting ofHsp90, Hsp70, Hsp25-27 and P-glycoprotein in hair follicles residing inskin or scalp that is exposed to the heat dose and that produces anincreased resistance of the hair follicles to chemotherapeutic drugs inthe scalp or other region susceptible to chemotherapy-induced alopeciaof a human patient or the skin of a mammalian; and b) administering achemotherapeutic drug to said human patient or said mammalian animal. 2.The method according to claim 1, wherein said administering the heatdose comprises heating hair follicles of the scalp of said human patientor the skin of said mammalian animal at about 39-45° C. for about 15-120minutes.
 3. The method according to claim 1, wherein the heat dose isadministered by a means selected from the group consisting of directcontact with heated surface or liquid, infrared radiation, microwaveradiation, ultrasound and radiofrequency radiation.
 4. The methodaccording to claim 3, wherein the heat dose is administered by directcontact with a heated surface.
 5. The method according to claim 3,wherein the heat dose is administered by direct contact with a heatedliquid.
 6. The method according to claim 3, wherein the heat dose isadministered by a infrared radiation.
 7. The method according to claim3, wherein the heat dose is administered by microwave radiation.
 8. Themethod according to claim 3, wherein the heat dose is administered byultrasound.
 9. The method according to claim 3, wherein the heat dose isadministered by radiofrequency radiation.
 10. The method according toclaim 2, wherein the heat dose is administered by a means selected fromthe group consisting of direct contact with heated surface or liquid,infrared radiation, microwave radiation, ultrasound and radiofrequencyradiation.
 11. A method for protecting a human patient or a mammaliananimal to be subjected to chemotherapy treatment of a tumor not residingin the scalp or other region susceptible to chemotherapy-inducedalopecia of the patient or the skin of the animal againstchemotherapy-induced alopecia, the protective method comprisingadministering a heat dose to the scalp or other region susceptible tochemotherapy-induced alopecia of the human patient or the skin of theanimal whereby hair follicles in the scalp or other region susceptibleto chemotherapy-induced alopecia of the patient or the skin of theanimal are heated to and maintained at a temperature of about 39-45° C.for about 15-120 minutes and administering a chemotherapeutic drug tosaid human patient or mammalian animal.
 12. The method according toclaim 11, wherein the heat dose is administered by a means selected fromthe group consisting of direct contact with heated surface or liquid,infrared radiation, microwave radiation, ultrasound and radiofrequencyradiation.
 13. The method according to claim 12, wherein the heat doseis administered by direct contact with a heated surface.
 14. The methodaccording to claim 12, wherein the heat dose is administered by directcontact with a heated liquid.
 15. The method according to claim 12,wherein the heat dose is administered by a infrared radiation.
 16. Themethod according to claim 12, wherein the heat dose is administered bymicrowave radiation.
 17. The method according to claim 12, wherein theheat dose is administered by ultrasound.
 18. The method according toclaim 12, wherein the heat dose is administered by radiofrequencyradiation.
 19. A method for protecting a human patient or a mammaliananimal to be subjected to chemotherapy treatment of a tumor not residingin the scalp or other region susceptible to chemotherapy-inducedalopecia of the patient or the skin of the animal againstchemotherapy-induced alopecia, the protective method comprisingadministering a heat dose to the scalp or other region susceptible tochemotherapy-induced alopecia of the patient or the skin of the animal,wherein the effective heat dose is a dose equal or greater to thatrequired to cause an increase in the concentration of a stress proteinselected from the group consisting of Hsp90, Hsp70, Hsp25-27 andP-glycoprotein in cells of hair follicles and administering achemotherapeutic agent to said human patient or said mammalian animal.20. The method according to claim 19, wherein the heat dose isadministered by a means selected from the group consisting of directcontact with heated surface or liquid, infrared radiation, microwaveradiation, ultrasound and radiofrequency radiation.